Fluid injection

ABSTRACT

The present invention generally relates to systems and methods for the control of fluids and, in some cases, to systems and methods for flowing a fluid into and/or out of other fluids. As examples, fluid may be injected into a droplet contained within a fluidic channel, or a fluid may be injected into a fluidic channel to create a droplet. In some embodiments, electrodes may be used to apply an electric field to one or more fluidic channels, e.g., proximate an intersection of at least two fluidic channels. For instance, a first fluid may be urged into and/or out of a second fluid, facilitated by the electric field. The electric field, in some cases, may disrupt an interface between a first fluid and at least one other fluid. Properties such as the volume, flow rate, etc. of a first fluid being urged into and/or out of a second fluid can be controlled by controlling various properties of the fluid and/or a fluidic droplet, for example curvature of the fluidic droplet, and/or controlling the applied electric field.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/220,847, filed Jun. 26, 2009, entitled “FluidInjection,” by Weitz, et al., incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

Research leading to various aspects of the present invention weresponsored, at least in part, by the National Science Foundation, GrantNos. DBI-0649865 & DMR-0820484. The United States Government has certainrights in the invention.

FIELD OF INVENTION

The present invention generally relates to systems and methods for thecontrol of fluids and, in some cases, to systems and methods for flowinga fluid into and/or out of other fluids.

BACKGROUND

The manipulation of fluids to form fluid streams of desiredconfiguration, discontinuous fluid streams, droplets, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. Dropletmicrofluidics are useful for a variety of purposes includinghigh-throughput analysis of chemical and biological systems.

In many applications, several fluids must be combined in a specificsequence. Existing methods describe achieving this result by separatelyemulsifying a plurality of fluids as droplets, and bringing the dropletsinto contact at which point the droplets may be coalesced to combine thefluids. While droplet coalescence has been demonstrated for pairs ofdroplets, it is difficult to control.

SUMMARY OF THE INVENTION

The present invention generally relates to systems and methods for thecontrol of fluidic droplets and, in some cases, to systems and methodsfor flowing a fluid into and/or out of other fluids. The subject matterof the present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In one aspect, a method is provided. The method, in this aspect,comprises providing a first fluid and a second fluid in contact with thefirst fluid at an interface, wherein the first fluid and the secondfluid do not substantially mix, and wherein at least one of the firstfluid and the second fluid is not a fluidic droplet contained within acarrying fluid. The method further comprises applying an electric fieldto the interface sufficient to disrupt at least a portion of theinterface and flowing at least a portion of the second fluid into thefirst fluid.

In another aspect, a method is provided that comprises providing a firstfluid and a second fluid in contact with the first fluid at aninterface, wherein the first fluid and the second fluid do notsubstantially mix, and wherein at least one of the first fluid and thesecond fluid is not a fluidic droplet contained within a carrying fluid.The method further comprises applying an electric field to the interfacesufficient to disrupt at least a portion of the interface and removingat least a portion of the second fluid into the first fluid.

In yet another aspect, a method is provided. The method, in this aspect,comprises applying an electric field to an interface defined between afirst fluid and a second fluid contained within a channel and applying apressure to the second fluid contained within the channel sufficient tocause at least a portion of the second fluid to enter the first fluid.

In one set of embodiments, the method includes acts of providing amicrofluidic system comprising a first microfluidic channel and a secondmicrofluidic channel contacting the first microfluidic channel at anintersection, providing a first fluid in the first microfluidic channeland a second fluid in the second microfluidic channel, and applying anelectric field to the interface to urge the second fluid to enter thefirst microfluidic channel. In some cases, the first fluid and thesecond fluid may contact each other at least partially within theintersection to define a fluidic interface. In certain instances, in theabsence of the electric field, the second fluid is not urged to enterthe first microfluidic channel.

The method, in another set of embodiments, includes acts of providing amicrofluidic system comprising a first microfluidic channel and a secondmicrofluidic channel contacting the first microfluidic channel at anintersection, providing a first fluid in the first microfluidic channeland a second fluid in the second microfluidic channel, and applying anelectric field to the interface to urge fluid from the firstmicrofluidic channel into the second microfluidic channel. In someembodiments, the first fluid and the second fluid may contact each otherat least partially within the intersection to define a fluidicinterface. According to some embodiments, in the absence of the electricfield, the first fluid is not urged to enter the second microfluidicchannel

In yet another set of embodiments, the method includes acts of providinga microfluidic system comprising a first microfluidic channel and asecond microfluidic channel contacting the first microfluidic channel atan intersection, providing a first fluid in the first microfluidicchannel and a second fluid in the second microfluidic channel, andurging the second fluid to enter the first microfluidic channel. Thefirst fluid and the second fluid may contact each other at leastpartially within the intersection to define a fluidic interface, atleast in some embodiments. In certain cases, when an electric field isapplied to the interface, the second fluid may be at least partiallyprevented from entering the first microfluidic channel.

The method, in still another set of embodiments, includes acts ofproviding a microfluidic system comprising a first microfluidic channeland a second microfluidic channel contacting the first microfluidicchannel at an intersection, providing a first fluid in the firstmicrofluidic channel and a second fluid in the second microfluidicchannel, and urging fluid from the first microfluidic channel into thesecond microfluidic channel. In some embodiments, the first fluid andthe second fluid may contact each other at least partially within theintersection to define a fluidic interface. In certain embodiments, whenan electric field is applied to the interface, the fluid can be at leastpartially prevented from entering the second microfluidic channel

In still another aspect, a microfluidic apparatus is provided. Theapparatus comprises, in one set of embodiments, a first fluidic channel,a second fluidic channel in fluidic communication with the first fluidicchannel at an intersection, and first and second electrodes positionedon generally opposing sides of the first fluidic channel or the secondfluidic channel, wherein the first fluidic channel, the second fluidicchannel, the first electrode, and the second electrode are positionedsuch that a plane intersects each of these.

In another set of embodiments, the microfluidic apparatus includes afirst microfluidic channel, a second microfluidic channel contacting thefirst microfluidic channel at an intersection, and first and secondelectrodes positioned on opposing sides of the first microfluidicchannel and the second microfluidic channel. In some embodiments, thesecond microfluidic channel connects to the intersection via an orificehaving an area of no more than about 90% of the average cross-sectionaldimension of the second microfluidic channel.

The microfluidic apparatus includes, in yet another set of embodiments,a first microfluidic channel, a second microfluidic channel contactingthe first microfluidic channel at an intersection, and first and secondelectrodes positioned on the same side of the first microfluidicchannel. In certain cases, the second microfluidic channel connects tothe intersection via an orifice having an area of no more than about 90%of the average cross-sectional dimension of the second microfluidicchannel.

In yet another aspect, an article is provided. The article comprises,according to one set of embodiments, a microfluidic channel comprising afirst plurality of droplets of a first droplet type and a secondplurality of droplets of a second droplet type distinguishable from thefirst droplet type, the first and second droplet types defining arepeating pattern within the microfluidic channel that repeats at leasttwice and has a repeat unit that includes more than a single droplet.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more applications incorporated by reference include conflictingand/or inconsistent disclosure with respect to each other, then thelater-filed application shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For the purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 shows a system for flowing fluid into and/or out of another fluidin accordance with an embodiment of the invention;

FIGS. 2A, 2B, and 2C show a droplet before injection, during injection,and after injection in accordance with another embodiment of theinvention;

FIG. 3 shows a system for flowing fluid into and/or out of another fluidin accordance with yet another embodiment of the invention;

FIGS. 4A-4B illustrate various embodiments of the invention comprisingpatterns of droplets of a first fluid and of a second fluid inaccordance with yet another embodiment of the invention;

FIGS. 5A-5B show a system for flowing fluid into and/or out of anotherfluid in accordance with still another embodiment of the invention;

FIG. 6 shows a plot of droplet size distribution in accordance with anembodiment of the invention;

FIGS. 7A-7B show plots of control of injection volume in otherembodiments of the invention;

FIGS. 8A-8B show fluid injection in accordance with another embodimentof the invention;

FIGS. 9A-9B show another embodiment of the invention for dropletinjection;

FIGS. 10A-10D illustrate electronic control of fluid injection inaccordance with yet another embodiment of the invention;

FIGS. 11A-11C illustrate droplet creation in accordance with stillanother embodiment of the invention;

FIGS. 12A-12C illustrate pressure measurements according to yet anotherembodiment of the invention; and

FIGS. 13A-13B illustrate a sensor in still another embodiment of theinvention.

DETAILED DESCRIPTION

The present invention generally relates to systems and methods for thecontrol of fluids and, in some cases, to systems and methods for flowinga fluid into and/or out of other fluids. As examples, fluid may beinjected into a droplet contained within a fluidic channel, or a fluidmay be injected into a fluidic channel to create a droplet. In someembodiments, electrodes may be used to apply an electric field to one ormore fluidic channels, e.g., proximate an intersection of at least twofluidic channels. For instance, a first fluid may be urged into and/orout of a second fluid, facilitated by the electric field. The electricfield, in some cases, may disrupt an interface between a first fluid andat least one other fluid. Properties such as the volume, flow rate, etc.of a first fluid being urged into and/or out of a second fluid can becontrolled by controlling various properties of the fluid and/or afluidic droplet, for example curvature of the fluidic droplet, and/orcontrolling the applied electric field.

One illustrative example is now provided with reference to FIG. 1. Itshould be understood, however, that other embodiments besides the oneillustrated in FIG. 1 are also contemplated in other embodiments of theinvention, as discussed in detail below. In the example of FIG. 1, asystem for flowing a first fluid (e.g., a fluid within a channel) intoand/or out of a second fluid (e.g., a fluidic droplet) is illustrated.FIG. 1 shows system 200 having a droplet source 210 from which droplets220 flow into first channel 230. Intersecting first channel 230 is fluidsource 235, which is used to control the flow of droplets in firstchannel 230, e.g., using flow-focusing techniques or other techniquessuch as is described in more detail below. Also as shown in FIG. 1 areelectrodes 250 and second channel 240, which intersects with firstchannel 230 at intersection 260. Electrodes 250 are positioned on oneside of first channel 230 near intersection 260, and opposite secondchannel 240. As the droplets flow past intersection 260, a fluidicinterface is formed between the droplets and an injectable fluid insecond channel 240. Electrodes 250 create an electric field that maydisrupt the interface between the droplet and the injectable fluid, thusallowing fluid to flow from the second channel into the droplet, therebyforming droplets 225. However, in the absence of an electric field, nodisruption of the interface may occur, and thus, fluid from secondchannel 240 does not enter into droplets 220. Droplets 225 containingthe injectable fluid subsequently leave intersection 260, e.g., flowinginto collection channel 270.

In certain aspects of the invention, systems such as those describedherein may be used to perform fluidic injections into and/or fluidicwithdrawals from a fluid, for example, from a channel containing afluid, from a fluidic droplet, or the like. As described in the exampleabove, these operations may be performed on a first fluid by disruptingan interface defined between a first fluid and a second fluid. It shouldbe understood that fluid injection and/or withdrawal may include only aportion of a fluid. In some cases, however, fluid exchange in bothdirections may occur; i.e., a portion of the first fluid may be injectedinto the second fluid while a portion of the second fluid may bewithdrawn into the first fluid. Likewise, disruption of an interface maycomprise disrupting a portion of the interface or the entire interface.It should also be understood that fluid exchange may comprise fluidinjection into and/or withdrawal from a fluid, depending on theembodiment.

Accordingly, the invention, in some aspects, relates to systems andmethods for fusing or coalescing two or more fluids into one fluid(e.g., fluid from a fluidic channel may be injected into a fluidicdroplet). For instance, in one set of embodiments, two or more fluidsmay be fused or coalesced into one droplet in cases where the two ormore fluids ordinarily are unable to fuse or coalesce, for example, dueto composition, surface tension, droplet size, the presence or absenceof surfactants, etc. For example, a fluid may be injected into one ormore fluidic droplets. In certain microfluidic systems, the surfacetension of the droplets, relative to the size of the droplets, may alsoprevent fusion or coalescence from occurring in some cases, absent thesystems and methods disclosed herein for fusing or coalescing fluids.Additional examples of fusing or coalescing fluidic droplets aredescribed in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al., incorporatedherein by reference.

In some embodiments, a first channel is in fluid communication with asecond channel at an intersection where the first channel and the secondchannel meet, e.g., as shown in FIG. 1. As shown in FIG. 1, the firstchannel may be designated as the “main” channel (channel 230) and thesecond channel may be designated as the “side channel” (channel 240).However, it should be understood that in other embodiments, otherchannel arrangements are possible, and that “main” and “side” channelsare for explanatory purposes only. Generally, as used herein, the “main”channel and the “side” channel may each contain fluid, and the mainchannel is the channel in which fluid flows when an electric field isturned off, with the interface separating fluid in the main channel fromfluid in the side channel, while the “side” channel holds fluid thatflows when the interface is disrupted. Typically, the “main” channel forcarrying a first fluid continues on past the intersection while the“side” channel for carrying a second fluid ends at the intersection,although the “main” channel need not be linear. However, the “main” and“side” channels need not have the T-arrangement shown in FIG. 1. Inother embodiments of the invention, for example, the side channel may bealigned with an outlet channel, the various channels may be joined in a“Y” arrangement or in a 3-dimensional arrangement, or the like. Anon-limiting example of a “Y” arrangement is shown in FIGS. 5A-5B.

In some cases, the first or main channel may have a cross-sectional area(i.e., defined perpendicular to fluid flow within the channel) that doesnot change substantially as the channel approaches the intersection. Inother instances, however, the cross-sectional area may increase ordecrease (i.e., constrict) as the first channel approaches theintersection. For example, the cross-sectional area of the first channelmay decrease approaching the intersection.

The second channel may intersect the first channel at an intersection atany point along the first channel. At the point of intersection betweenthe first channel and the second channel, the second channel may meetthe first channel via an orifice in some embodiments, i.e., a portion ofthe second channel at the intersection with the first channel may have across-sectional area that is smaller than the cross-sectional area ofthe second channel leading up to the intersection. In other embodiments,however, no orifice may be present, or the cross-sectional area of thesecond channel may even be relatively larger in some cases. The orificemay be of any shape, for example circular, elliptical, triangular,rectangular, etc., and may be positioned in any suitable position in thesecond channel, e.g., at an end or on a side of the second channel, suchas is shown in FIG. 9.

As described herein, the geometry of the intersection may affect fluidexchange between a fluid in the first channel and a fluid in the secondchannel. The orifice may have an average cross-sectional dimension lessthan about 100 microns, less than about 30 microns, less than about 10microns, less than about 3 microns, less than about 1 micron, less thanabout 300 nm, less than about 100 nm, less than about 30 nm, less thanabout 10 nm, etc. In some cases, the orifice may have a cross-sectionalarea that is no more than about 90% of the average cross-sectionaldimension of the channel, and in some cases, the area is no more thanabout 80%, no more than about 70%, no more than about 60%, no more thanabout 50%, no more than about 40%, no more than about 30%, no more thanabout 20%, or no more than about 10% of the average cross-sectionaldimension of the channel. The orifice may be flush with an intersectingwall of the first channel (i.e., the orifice may be defined by a lack ofa portion of a wall of the first channel). One non-limiting example isshown in FIG. 2. Alternatively, in some cases, the orifice may be in aposition that protrudes into the first channel.

In some embodiments, one or more electrodes are provided that can applyan electric field to an intersection of a first channel and a secondchannel. In some cases, the electrodes may be positioned such that theelectrodes create an electric field maximum that contains theintersection, or at least is proximate the intersection. For instance,the electrodes may be positioned to create an electric field or anelectric field maximum located where the second channel intersects withthe first channel, or such that the interface between two fluids in thechannels experiences a suitable electric field. The electrodes may bepositioned relative to the channels in a variety of configurations. Insome examples, an electrode may be positioned essentially opposite asecond channel. Alternatively, an electrode may be positionedsubstantially to one side of the second channel. In some embodiments, anelectrode may be positioned above or below the second channel (e.g., inanother layer of the device).

In some cases, a plurality of electrodes are provided. For example, twoelectrodes may be positioned essentially on the same or opposite sidesof the first or second channels. In some instances, two electrodes maybe positioned on opposite sides of both the first channel and secondchannel. An example of an embodiment in which the electrodes arepositioned on the same side is shown in FIG. 1, while an embodiment inwhich the electrodes are positioned on opposite sides is shown in FIG.9. In some embodiments, a first electrode and a second electrode may bepositioned such that a plane intersects both electrodes. In some cases,the electrodes may be positioned to be co-planar with one or morechannels (e.g., as shown in FIG. 1), e.g., such that the first fluidicchannel, the second fluidic channel, and the electrodes are positionedsuch that a plane intersects each of these.

Referring again to FIG. 9, FIG. 9A shows a photomicrograph of an examplesystem, while FIG. 9B is a schematic diagram of the same system. In thisexample, system 700 includes a first electrode 710, and an opposed,second electrode 720 that is co-planar with first electrode 710. In theexample in FIG. 9B, during use, electrode 710 is positive, whileelectrode 720 is negative. However, in other embodiments, electrode 710may be negative and electrode 720 may be positive. In this figure,electrodes 710 and 720 are positioned on opposite sides of intersection750 between a first, main channel 730 and a second, side channel 740.The electrodes may be positioned such that the electric field createdbetween the electrodes is centered over the intersection between firstchannel 730 and second channel 740, as is shown in FIG. 9B, or theelectrodes may be positioned in an off-center configuration, in otherembodiments of the invention. As shown here, electric field lines 715are generated going from the positive electrode 710 to the negativeelectrode 720. Due the location of the electrodes, electric field lines715 passing from electrode 710 to electrode 720 pass over intersection750. Thus, by controlling the voltage between the electrodes, theelectric field at intersection 750 may be controlled. Also shown in FIG.9B, first channel 730 is filled with a first fluid, while second channel740 is filled with a second fluid (shown here as a different shade forpurposes of clarity). As discussed herein, the second fluid from secondchannel 740 may be injected into first channel 730, and/or into droplets(not shown) within the first channel 730.

An example of use of such a system is now discussed with reference toFIG. 8B. In this figure, fluid within a first channel 750 passes byfluid extending from second channel 760, and through the controlledapplication of an electric field (e.g., by electrodes, not shown in FIG.8), a portion of the fluid from second channel 760 is injected into adroplet 770 contained within first channel 750 as droplet 770 passesthrough intersection 765 of first channel 750 with second channel 760.As previously discussed, the amount of fluid injected into the dropletmay be controlled by controlling the strength of the electric fieldapplied to the intersection, and/or other factors such as is describedherein. For example, in one set of embodiments, if no electric field isapplied via the electrodes, then no fluid injection may occur, or thevolume of fluid that is injected into first channel 750 from secondchannel 760 may be controlled, at least in part, by controlling theelectric field applied to the intersection.

An electrode as discussed herein may be fabricated from any suitablematerial, and if two or more electrodes are present, the electrodes maybe formed from the same or different materials. Non-limiting examples ofelectrode materials include metals, metalloids, semiconductors,graphite, conducting polymers, and the like. The electrode may have anyshape suitable for applying an electric field. In some cases, anelectrode may have an essentially rectangular shape. As another example,an electrode may be elongated and have a tip defined as a region of theelectrode closest to an intersection between a first channel and asecond channel. In some embodiments, the tip of an electrode may have awidth that is similar to a width of the second channel. In otherembodiments, the tip of an electrode may have a width substantiallylarger than a width of the second channel. The electrode shape can beflat, V-shaped, or any other suitable shape, such as the shapesdiscussed herein.

In some cases, the tip of the electrode is constructed such that anelectric field maximum is created, e.g., in the intersection orproximate the intersection, as previously discussed. For example, anelectrode may be constructed such that the electric field gradient isoptimized in the direction of the desired interface disruption. In someinstances, e.g., where multiple electrodes are used (e.g., as discussedrelative to FIG. 3 herein), the electrodes may be constructed tominimize interference between one or more electrodes and one or morechannels; for example, by minimizing the unintended exposure of a firstinterface to an electric field by an electrode intended to expose asecond interface positioned in a different location than the firstinterface to an electric field. In some embodiments, reducing the sizeof an electrode tip can allow more focused application of an electricfield by the electrode tip such that one or more interfaces are notunintentionally exposed to the electric field, and/or are exposed torelatively lower electric field strengths. This may be advantageous, forexample, in instances where it is desired to reduce the distance betweena plurality of injection systems.

The electric field produced by the electrodes, in some embodiments, isgenerated using an electric field generator, i.e., a device or systemable to create an electric field that can be applied to the fluid, e.g.,via one or more electrodes. For example, the electric field generatormay include a voltage source and one or more electrodes. Voltage sourcesinclude batteries, wall current, fuel cells, or the like, and a widevariety of voltage sources are commercially available. The electricfield generator may produce an AC field (i.e., one that variesperiodically with respect to time, for example, sinusoidally, sawtooth,square, etc.), a DC field (i.e., one that is constant with respect totime), a pulsed field, etc. The electric field generator may beconstructed and arranged to create an electric field within a fluidcontained within a channel or a microfluidic channel. The electric fieldgenerator may be integral to or separate from the fluidic systemcontaining the channel, according to some embodiments. As used herein,“integral” means that portions of the components integral to each otherare joined in such a way that the components cannot be manuallyseparated from each other without cutting or breaking at least one ofthe components. In addition, in some cases, the electric field may beautomatically controlled, e.g., by aid of a computer or an automaticdevice.

As mentioned, in some instances, the electric field is applied to anintersection between a first channel and a second channel; for example,the electric field may be applied to an intersection between a firstchannel and a second channel. The electric field may be appliedcontinuously, periodically, or intermittently, depending on theembodiment, and may be AC, DC, etc. For example, while an intersectionis exposed to an electric field, a fluidic droplet may be urged into orthrough the intersection. The electric field may be applied to disruptthe interface formed between the fluidic droplet and, for example, afluid in the second channel, e.g., as discussed above, thereby allowingfluid exchange from the second channel into the first channel to occur.As another example, by controlling the electric field, fluid from thesecond channel may be urged into the first channel to create one or morenew droplets contained within the first channel.

The voltage applied to the electrodes may be any suitable voltage fordisrupting a fluidic interface. For example, the voltage may be between0.1 V and 10,000 V, between 0.1 V and 1,000 V, between 0.1 V and 300 V,between 0.1 V and 100 V, between 0.1 V and 50 V, between 0.1 V and 30 V,between 0.1 V and 10 V, or the like. As applied voltage may be appliedcontinuously, pulsatile, intermittently, randomly, etc. The pulses maybe DC, or AC, with any suitable frequency, for example, frequencies inthe hertz, kilohertz, or megahertz ranges, etc.

For example, in one embodiment, the electric field that is applied maybe pulsed. For instance, the electric field may be applied when afluidic droplet is present at an intersection, but not applied at othertimes. In another embodiment, the electric field is applied while afluidic droplet is in front of the second channel. For example, theelectric field may be applied for a period of time sufficient forinjection and/or withdrawal of a specific volume of fluid, e.g., theelectric field may on for a period of time and off for a period of timewhile the fluidic droplet is in front of the second channel.

By applying a voltage across the electrodes, e.g., via an electric fieldgenerator, an electric field may be created, which may be modeled aselectric field lines passing from the positive electrode to the negativeelectrode, as is shown in FIG. 9B. The electric field lines may passthrough the interface between the fluids, for example, in the samedirection as the flow during injection of a fluid from the secondchannel into the first channel. Other angles may be used in otherembodiments. The electrodes may be positioned to center the electricfield over the intersection, or offset from the center, for example, togenerate a component of the electric field in a direction of a flowchannel.

In some aspects of the invention, as described, techniques for injectingand/or withdrawing a first fluid from a second fluid involve formationof an interface between the first fluid and the second fluid, disruptionof the interface (e.g., using an electric field) such that it does notpresent a barrier between the first and second fluids, and allowstransfer of fluid between the first fluid and the second fluid. Forinstance, when an interface between a first fluid and a second fluid(e.g., between a fluid within a second channel and a droplet in a firstchannel positioned at an intersection with the second channel) isdisrupted, fluid may flow from the first fluid to the second fluid, fromthe second fluid to the first fluid, or both (e.g., through diffusion orconvection of the two fluids with respect to each other). As an example,by controlling the difference in pressures between the first and secondchannels, fluid may be urged to flow preferentially in one direction oranother (e.g., towards the droplet, i.e., injection, or away from thedroplet, i.e., withdrawal).

Thus, in certain cases, at least a portion of an interface between twofluids may be disrupted to allow fluid flow between the two fluids tooccur. In some embodiments, at least a portion of the interface may bedisrupted by electric field, e.g., applied by using electrodes such asthose described herein. The electric field may be, for example, an ACfield, a DC field, a pulsed field, etc.

Without wishing to be bound by any theory, it is believed thatapplication of an electric field causes forces that are acting todisrupt the interface (e.g., shear forces, mechanical forces, electricalforces such as Coulombic forces, etc.) to become larger than forcesacting to maintain the interface (e.g., surface tension, surfactantmolecule alignment and/or steric hindrance, etc.). For example, onepossible mechanism by which the electric field disrupts the fluidicinterface is that dipole-dipole interactions induced by the electricfield in each of the fluids may cause the fluids to become electricallyattracted towards each other due to their local opposite charges, thusdisrupting at least a portion of the interface and thereby causing afluidic connection to form between the fluids, which may be used forinjection, withdrawal, mixing, or the like. Another possible mechanismby which the electric field disrupts the interface is that the electricfield may produce a force directly on the fluids, thereby resulting incoalescence.

For instance, in one set embodiments, the fluid to be injected into achannel may be charged directly, for example, by application of anelectric field using electrodes positioned to apply the electric fieldto or proximate the intersection. As a specific example, as a dropletwithin a first channel approaches the intersection of the first channelwith the second channel, the droplet may at least partially polarize,e.g., as is depicted in FIG. 8A. The polarization may generate anattractive force between the fluids, which may cause them to becomeattracted to each other, and to merge in some instances. In some cases,the fluids may be at least partially conducting, and the fluids may besurrounded or contained within an insulating fluid, for example, oil,which may allow the fluids to exhibit charge or charge separation withinthe applied electric field.

In some embodiments, the fluidic interface may be disrupted after acertain threshold electric field strength has been reached. Thethreshold electric field strength may be any minimum value able todisrupt the interface, and may vary by application. For example, factorssuch as the viscosity or density of the fluids contained within thechannel, the flow rate of fluids within the channels, the geometry(e.g., sizes or dimensions) of the channels, the angle at which thechannels meet at an intersection, the presence of other forces appliedto the fluid, etc., may affect the threshold electric field strength fora particular application. Non-limiting examples of electric fieldstrength for disrupting an interface include electric field strengthsgreater than about 0.01 V/micrometer, greater than about 0.03V/micrometer, greater than about 0.1 V/micrometer, greater than about0.3 V/micrometer, greater than about 1 V/micrometer, greater than about3 V/micrometer, greater than about 10 V/micrometer, etc. It should beunderstood that values outside these ranges may also be used in someinstances. In some cases, the amount of fluid transfer may beessentially constant as a function of voltage above the thresholdvoltage. In other embodiments, the amount of interface disruptiongenerally increases as electric field strength increases.

Non-limiting example of control and disruption of the interface follows.In general, control of the interface depends on factors such as thenature of the fluids within the channels (e.g., their viscosity ordensity), the curvature of the fluids at the fluidic interface, the sizeof the channels (which affects their curvature at the interface), thesize of the intersection of the channels, or the like. An example of thecontrol and disruption of an interface between two fluids is nowdescribed with reference to a system having a first channel and a secondchannel that meet at an intersection, where the first (“main”) channelcarries a fluidic droplet and a second (“side”) channel carries a fluidto be injected into the fluidic droplet, and/or is used to withdrawfluid from the fluidic channel.

As one non-limiting example, a fluidic droplet in a first channel mayform a fluidic interface with a fluid in a second channel, e.g., whenthe first channel and the second channel meet at an intersection. Insome cases, the fluidic droplet in the first channel may be sufficientlylarge that the fluidic droplet is in contact with the walls of the firstchannel; in some cases, the width of the first channel may affect theradius of curvature of the fluidic droplet. The radius of curvature offluid in the second channel at the interface of the fluid with the firstchannel may be controlled at least in part, in certain embodiments, bythe cross-sectional area of the second channel at the intersection withthe first channel (e.g., at an orifice or nozzle of the second channel,if one is present, as it contacts the first channel). That is, as thecross-sectional area of an orifice or intersection between the first andsecond channels decreases, the radius of curvature of the fluid in thesecond channel as it contacts the fluid in the first channel decreases.In some cases, the radius of curvature of the fluid in the secondchannel may be defined, at least in part, by pressurizing the fluidwithin the second channel such that the fluid at least partiallyprotrudes from the orifice or interface into the first channel.

As a non-limiting example of this, referring to FIG. 2A, fluidic droplet300 is shown in first channel 310 flowing from left to right in thefigure. Fluid 320 is also shown in this example protruding from a secondchannel 330, where the second channel has orifice 340. It should benoted that, as is shown in FIG. 2, the orifice is at the end of atapered portion of second channel 330; the orifice need not have thesame size or average diameter as the fluidic channel flowing into theorifice. In this example, the radius of curvature R₁ of the fluidicdroplet contained within first channel 310 is larger than the radius ofcurvature R₂ of the fluid entering first channel 310 from second channel330. The fluidic droplet and fluid are thus in contact with each otherforming an interface 350, as is shown in FIG. 2A.

In FIG. 2B, the interface between the fluidic droplet and the enteringfluid may be disrupted by exposure of the interface to a suitableelectric field, as is discussed herein. It is believed, without wishingto be bound by any theory, that application of an electric field mayalter the dipole moments of the fluids at the interface between thefluids, which may be at least sufficient to break the surface tension ofthe interface separating the fluids, thereby disrupting the interfaceseparating the fluids and allowing fluid exchange to occur. In somecases, as discussed herein, the size and/or shape of the interface mayalso be controlled by controlling the electric field at the interfacebetween the fluids; for example, stronger electric fields may increasealteration of the dipole moments of the fluids at the interface betweenthe fluids, which may thereby alter the amount of disruption, thethreshold of disruption, and/or the amount of fluid able to be exchangedbetween the fluidic droplet and the entering fluid. It should also benoted that fluidic droplet 300 is used by way of example only; in otherembodiments, fluid from second channel 330 may be injected directly intofirst channel 310, i.e., without the presence of fluidic droplet 300within first channel 310. In some cases, this may cause the creation ofnew fluidic droplets within the first channel.

The direction of fluid exchange between the fluidic droplet and fluidfrom second channel 330 may be controlled, according to variousembodiments of the invention, by controlling factors such as the fluidicpressures of the various fluids, the strength of the electric field, theshape of the channel, the nature of the channel intersection, or thelike. As a specific non-limiting example, in some cases, fluidicexchange may be controlled by controlling the pressures or relativepressures of the fluids. For instance, and without wishing to be boundby any theory, it is believed that by controlling the radii of curvatureof the fluids such that R₁ (in the first channel) is greater than R₂ (inthe second channel), for instance by controlling the pressure of thefluidic droplet (p₁) to be less than the pressure of the fluid in thesecond channel (p₂), fluid may flow from the second channel into thefluidic droplet. Thus, the fluid in the second channel can flow or be“injected” into the fluidic droplet after disruption of the interface inthis example. In other cases, however, e.g., as discussed below, thepressures may be controlled such that R₂ is greater than R₁, and fluidmay instead be withdrawn from the fluidic droplet into the secondchannel.

As mentioned, the interface between the fluids may be controlled bycontrolling the pressure (or relative pressure) and/or the electricfield applied to the interface. In certain embodiments, control of theelectric field may afford electronic control of the interface, e.g.,whether or when to control injection and/or withdraw of fluid into achannel and/or into a fluidic droplet within a channel. For example, bymodulating the electric field, the position of the interface between thetwo fluids may be adjusted. As an example, the position of the interfacemay be relatively higher when an electric field is applied, as is shownin FIG. 10B, compared to when there is no electric field (or a weakerelectric field), as is shown in FIG. 10A. The position of the interfacemay also be controlled dynamically in some embodiments, for example, bycontrolling the voltage applied to the electrodes.

As a specific non-limiting example, referring now to FIG. 10C, when thevoltage applied to the electrodes is adjusted, thereby adjusting thestrength of the electric field created between the electrodes that isapplied to the fluidic interface, the location of the interface respondscorrespondingly, as is shown in FIG. 10D (showing the position of theinterface in this non-limiting example). (It should be noted that bothFIGS. 10C and 10D are on the same time scale). Thus, by controlling theelectric field applied to the interface, the position of the interfacecan likewise be controlled. As mentioned, in some embodiments, theelectric field may be controlled by controlling the voltage applied tothe electrodes disposed about the interface, e.g., via controlling theintensity or polarity of the voltage, and/or (e.g., if AC), controllingthe amplitude, frequency, etc. of the voltage. For instance, by usinghigher voltages, the interface between the fluids may be extendedfarther into the first channel, allowing a larger volume to be injectedinto a fluidic droplet, whereas a smaller voltage may be used to causethe interface to extend less far into the channel, causing a lesseramount of fluid to be injected into the droplet. The interface positioncan also be modulated, in some embodiments, at suitable frequencies, forexample, at frequencies that are comparable to the rate of dropletformation or passage by the interface, thereby allowing the amount ofvolume to be injected into each droplet to be individually controlled.

In addition, in certain embodiments, if the electric field is increasedor controlled sufficiently, the fluidic interface may be extended into afirst channel so far that the fluid from a second channel breaks offinto the first channel to form a new droplet. An example of this processis discussed with reference to FIGS. 11A and 11B. In FIG. 11A, when noelectric field (or an insufficient electric field) is applied to aninterface 930 created at an intersection between first channel 910 andsecond channel 920, the interface between a fluid within the secondchannel and fluid within the first channel is not disrupted, and thus,fluid within the second channel does not readily flow into the firstchannel, i.e., droplet creation is “off.” However, in FIG. 11B, under anapplied electric field (represented by electric field lines 950) createdusing electrodes 915, the interface between the fluids can be extendedinto the first channel.

Under suitable conditions (for example, a relatively rapid rate of fluidflow within the first channel), interface 930 may be extended quite farinto first channel 910, e.g., as is shown in FIG. 11B. In some cases,the interface extends so far that the interface is disrupted, causingsome fluid within second channel 920 to enter first channel 910 as adiscrete droplet. Repeating this process may be used to create aplurality of fluidic droplets within first channel 910. It should benoted that, since this process is controlled electronically, rapiddroplet production may be achieved in some cases. For instance, at leastabout 10 droplets per second may be created in some cases, and in othercases, at least about 20 droplets per second, at least about 30 dropletsper second, at least about 100 droplets per second, at least about 200droplets per second, at least about 300 droplets per second, at leastabout 500 droplets per second, at least about 750 droplets per second,at least about 1000 droplets per second, at least about 1500 dropletsper second, at least about 2000 droplets per second, at least about 3000droplets per second, at least about 5000 droplets per second, at leastabout 7500 droplets per second, at least about 10,000 droplets persecond or more droplets per second may be created in such a fashion.Accordingly, in some embodiments, by controlling the electric field,individual fluidic droplets may be created within a channel. Inaddition, by adjusting factors such as the voltage, flow rates, and/orpressures within the fluids, the volume of the droplet thus formedwithin the channel may be controlled.

In another set of embodiments, the use of the electric field may bereversed, where the application is used to turn off or decrease dropletcreation, instead of being used to create droplets. For example, in somecases, a fluidic droplet system such as those discussed herein may beused to create droplets. The fluidic droplet system may create droplets,e.g., using flow-focusing techniques or other techniques such asdisclosed herein. When no electric field is applied, droplets may beformed; however, when a sufficient electric field is applied, e.g.,using electrodes, the fluidic interface may be contracted, which mayalter or inhibit droplet formation.

For example, in FIG. 11C, at an interface between a first fluid withinfirst channel 910 and a second fluid within second channel 920, dropletsmay be created in the absence of an electric field, e.g., due to motionor pressures within the second channel that urge the second fluid intothe first channel, e.g., forming droplets (for example, if the first andsecond fluids are substantially immiscible). However, upon theapplication of suitable electric field, as is shown in this figure withelectric field lines 950 between electrodes 915, the applied electricfield cause interface 930 between first channel 910 and second channel920 to move; in this case, the interface moves “upstream” into secondchannel 920 and away from first channel 910, thereby slowing orinhibiting droplet formation of the second fluid into the first fluidwithin first channel 910. Accordingly, in another set of embodiments,the application of an electric field may be used to partially orcompletely prevent the second fluid from entering the first fluid. Inaddition, an analogous system may be used to prevent fluid from thefirst channel from being withdrawn into the second channel; i.e., in theabsence of an electric field (or an insufficient electric field), fluidmay be withdrawn into the second channel from the first channel, butupon application of a suitable electric field, the interface between thefluids moves such that fluid cannot be withdrawn into the second channelfrom the first channel.

In some aspects, fluid may be injected into a fluidic channel, e.g., ina fluidic droplet contained within the channel, which may in some casescause mixing of the injected fluid with other fluids within the fluidicdroplet to occur. The present invention broadly contemplates, in certainembodiments, various systems and methods for injecting fluid, e.g., intoa fluidic droplet. It should be understood that, in the descriptionsherein involving the “injection” of a fluid from a second channel into afirst channel, the fluid that is injected may be injected into a dropletcontained within the first channel and/or into fluid contained withinthe first channel, e.g., forming a new droplet. Thus, in someembodiments, fluid injection using a first channel, a second channel,and electrodes as discussed herein may be used to create new droplets ofa fluid from the second channel that are individually contained withinfluid within the first channel.

For example, in one set of embodiments, the fluid may be injected intothe fluidic droplet using a needle such as a microneedle, a nozzle, anorifice, a tube, or other such devices. In another set of embodiments,the fluid may be injected directly into a fluidic droplet using afluidic channel as the fluidic droplet comes into contact with thefluidic channel. For instance, in certain embodiments, a first channelcontaining a fluid may be intersected by a second channel at anintersection. Fluid from the second channel may be injected into thefirst channel, for example, using suitable pressures within one or bothof the channels, e.g., a pump. For example, when a droplet containedwithin the first channel passes through the intersection, fluid from thesecond channel may be urged into the intersection, thereby entering thedroplet and causing injection of fluid from the second channel into thedroplet to occur.

The amount of fluid injected can be controlled, in certain embodiments,by controlling properties such as the relative pressure of the fluids,the residence time of a droplet in the intersection, the viscosity ofthe fluids, the electric field applied to the interface, etc. Forexample, referring now to FIGS. 2B and 2C, fluidic droplet 300 is influidic communication with fluid from a second channel 330, and thusfluid can flow from second channel 330 into droplet 300. However, asfluidic droplet 300 moves through first channel 310, shear betweenfluidic droplet 300 and fluid from second channel 330 increases, and maycause the fluidic droplet to detach from the second channel 330 (FIG.2C). In some cases, the interface between the fluid in first channel 310and the fluid in second channel 330 may be restored by the shearingaction of droplet flow past the intersection, as is shown in FIG. 2C.

The volume of fluid that may be injected and/or withdrawn may be anysuitable amount, depending on the embodiment. For instance, the volumeinjected and/or withdrawn may be less than about 10 microliters, lessthan about 1 microliter, less than about 100 nanoliters, less than about10 nanoliters, less than about 1 nanoliter, less than about 100picoliters, less than about 10 picoliters, less than about 1 picoliter,etc. In some cases, fluid may be injected and/or withdrawn while thefluid in the first channel is in motion (i.e., flowing through a firstchannel). In other cases, fluid may be injected and/or withdrawn whilethe fluid in the first channel is held stationary. For example, pressurein the first channel may be controlled such that a droplet is urged toan intersection between the first channel and a second channel. Thepressure and/or fluid flow within the first channel may then bedecreased such that the droplet is then held stationary at theintersection, thereby allowing a desired amount of fluid to be injectedand/or withdrawn into the droplet. The pressure may then be increasedand/or fluid flow may be controlled to urge the droplet away from theintersection once a desired amount of fluid has been transported.

In some instances, the second channel may be configured, e.g., with apump or other pressure control device such that fluid can be forciblyinjected and/or withdrawn from the first channel, e.g., to or from afluidic droplet, for example, without reliance on a difference in radiiof curvature of the interface, or the like.

The flow velocity of the fluidic droplet within the first channel may bedetermined in some embodiments by factors such as the pressure or thepressure difference between the fluidic droplet in the first channel andthe fluid in the second channel, the fluid pressure in one or bothchannels, the size of the orifice between the first channel and thesecond channel, the angle of intersection between the first and secondchannels, etc. as discussed above. The fluid pressure may be controlledusing any suitable technique, for example, using a pump, siphonpressure, or the like.

As mentioned, the volume of fluid injected and/or withdrawn may becontrolled using any suitable technique, for example, by controlling thepressures of the various fluids, the volumetric flow rates, the strengthof the applied electric field, or the like. For instance, in someembodiments, the flow rate of fluid in the first channel can be used tocontrol the volume of fluid injected and/or withdrawn. It is believedthat this can be controlled since the flow rate of fluid in the firstchannel controls the flow rate of fluidic droplets in the first channel,which thereby controls the amount of time that the fluidic droplets arepresent at the intersection and/or exposed to fluid in the secondchannel.

In another set of embodiments, the pressure and/or the different inpressures between the fluids may be used to control the volume of fluidinjected and/or withdrawn. For example, by equalizing the pressures,flow between the fluids may be minimized, while fluid may be urged toflow into or from a droplet or a channel by a suitable difference inpressures between the fluids. For example, the pressure in the secondchannel may be increased, relative to the first channel, to cause fluidflow to occur into the first channel, e.g., into a droplet within thefirst channel.

In some embodiments, the volume of fluid exchanged between two fluidsmay be controlled by controlling the residence time of a first fluid inproximity to a second fluid, e.g., by controlling the residence time ofa fluid in the first channel and positioned in front of a secondchannel. As a non-limiting example, the residence time of a fluidic in adroplet in the first channel positioned in front of the second channelmay be controlled by varying the flow rate of fluid in the firstchannel. That is, a longer residence time may be achieved by slowing theflow rate or even stopping the flow of the fluid in the first channel,relative to the second channel. Likewise, a shorter residence time maybe achieved by increasing the flow rate of the fluid in the firstchannel.

In another set of embodiments, the duration of the electric fieldapplied, e.g., while a droplet is positioned in an intersection of firstand second channels may by varied. For example, to allow more fluid toexchange between fluid in the first channel and in the second channel,the interface may be disrupted for a longer period of time. To allow asmaller amount of fluid exchange, the interface may be disrupted for ashorter period of time.

In one set of embodiments, the systems and methods described herein maybe used to transfer fluid between a first channel and a second channel.For example, a first fluid in a first channel may form an interface witha second fluid in a second channel. Disruption of the interface betweenthe first fluid and the second fluid may allow fluid to be exchangedbetween the two fluids, as shown in FIGS. 5A-5B. In FIG. 5A, a firstchannel 600 containing a first fluid (the direction of flow is indicatedby the arrows in the channel) is connected to a second channel 610having a second fluid that is stationary. The two fluids are separatedby an interface 620 when electrodes 630 are in the “OFF” state (orotherwise are at a voltage insufficient to disrupt interface 620). InFIG. 5B, electrodes 630 are in the “ON” state (i.e., at a voltage atleast sufficient to disrupt interface 620), thereby causing disruptionof interface 620 and allowing the second fluid in second channel 610 toflow into first channel 600. In some cases, the flow of fluid betweenthe two channels may be facilitated, for example, by applying pressureto the first or second channel, reducing pressure on the first or secondchannel etc. As described above, the interface may be disrupted usingmethods such as exposure of the interface to an electric field.

As another example, the geometry of a second channel intersecting afirst channel, in some cases, may influence fluid injection and/orwithdrawal. Without wishing to be bound by any theory, it is believedthat such control may be achieved since the flow velocity of the fluidin the second channel into a fluidic droplet is generally inverselyrelated to the hydrodynamic resistance of the second channel. Thisproperty may be dominated, in some instances, by the narrowestconstriction of the second channel. The flow velocity may be controlled,for example, by selecting appropriate channel dimensions and/or bycontrolling the pressures of the fluids within the channel. In someembodiments, for instance, the flow velocity may be less than about 1mm/second, less than about 100 microns/second, less than about 10microns/second, less than about 1 micron/second, less than about 100nm/second, less than about 10 nm/second, less than about 1 nm/second,etc.

In certain embodiments, as mentioned, a second channel may be used forwithdrawing fluid from a fluid in a first channel to which the secondchannel intersects. In some cases, the fluid in the first channel may becontrolled to have a higher pressure than the fluid within the secondchannel. This may be accomplished in a variety of ways. In someinstances, for example, the radii of curvature of a fluidic droplet inthe first channel and a fluid in the second channel may be controlledsuch that the radius of curvature of the fluidic droplet is smaller thanthe radius of curvature of the fluid in the second channel thatinterfaces with the first channel. For example, the fluidic droplet maybe controlled within the first channel resulting in a fluidic dropletwith a smaller radius of curvature. In another embodiment, thecross-sectional area of the second channel (or the cross-sectional areaof an orifice of the second channel) may be increased such that theradius of curvature of fluid emerging from the second channel into thefirst channel is larger, relative to a droplet in the first channel.

In some cases, the interior of a channel may be modified to change thewettability of the channel. For example, a channel such as a secondchannel may be modified such that the fluid emerging from the secondchannel into a first channel can adopt negative curvature inside thesecond channel (i.e., a concave configuration, concave being defined aswithdrawn into the second channel as opposed to protruding from thesecond channel into the first channel). In such instances, for example,the second channel may be able to withdraw fluid rapidly from a fluid inthe first channel, e.g., a droplet contained within the first channel.Examples of techniques for controlling or altering the hydrophilicity orhydrophobicity of a surface are disclosed in International PatentApplication No. PCT/US2009/000850, filed Feb. 11, 2009, entitled“Surfaces, Including Microfluidic Channels, With Controlled WettingProperties,” by Abate, et al., incorporated herein by reference in itsentirety.

In some embodiments, a system may include multiple channels and/orelectrodes. Such a system, for example, may be used to perform multipleinjection/withdrawal operations, e.g., in series. For instance, FIG. 3depicts a time sequence demonstrating injection of fluids into a fluidicdroplet. In this figure, a plurality of injection systems are arrangedin series. Each of the injection systems may contain the same, or adifferent fluid to be injected into a channel. For example, in certaininstances, a single fluidic droplet may be injected with a plurality ofdifferent fluids as it flows through a channel, e.g., by use of aplurality of injection systems. As another example, in some embodiments,a first set of fluidic droplets may be injected with a first fluid and asecond set of fluidic droplets may be injected with a second fluid.

As a specific non-limiting example, FIG. 3 shows electrodes 400, 405,and 410, with electrodes 400 and 405 in the “off” state (e.g., zeroelectric field, or an electric field that is below a threshold level atleast sufficient to disrupt an interface). Electrode 410 is in the “on”state, applying an electric field sufficient to disrupt an interface. Afluidic droplet 420, having moved past channels 430 and 432 containing acolored fluid, has not been injected with colored fluid since electrodes400 and 405, which can apply an electric field at the intersection ofchannel 435 and channels 430 and 432, respectively, are in the “off”state. Fluidic droplet 420 is, however, injected with colored fluid fromchannel 434 while at the intersection of channel 435 and channel 434since electrode 410 is in the “on” state.

Other arrangements for performing multiple injections will be apparentto those skilled in the art. Multiple injections may be used togenerate, for example, a combinatorial library of fluidic droplets,where each fluidic droplet has a unique combination of injectionsdifferentiated by properties such as injection content, injection amount(i.e., concentration, volume, etc.), time of injection (i.e., atdifferent periods of time), etc.

Another aspect of the invention generally relates to a plurality ofdroplet types contained within a channel, such as a microfluidicchannel. By using systems such as those described herein, droplets maybe controlled within a channel to be distinguishable, for instance, onthe basis of color, size, a species contained within some of thedroplets, or the like. Thus, as a specific example, some droplets of aplurality of droplets may be injected to create a first plurality ofdroplets of a first droplet type and a second plurality of droplets of asecond droplet type distinguishable from the first droplet type, forinstance, by using a dye. Other examples of potentially suitabledistinguishing characteristics include composition, concentration,density, etc.; optical properties such as transparency, opacity,refractive index, etc.; or electrical properties such as capacitance,conductance, resistivity, etc.

In one set of embodiments, using the systems and methods describedherein, any arbitrary pattern of droplets within a channel may beinjected, e.g., arbitrary, random, encoding a repeating sequence, or thelike. For instance, the droplets may be injected in an alternatingfashion (e.g., between first and second droplet types), or as is shownin FIGS. 4A and 4B, the fluidic droplets may be injected in longerrepeating or arbitrary patterns, containing any number of dropletswithin a repeat unit (which is repeated at least twice within thechannel to produce the repeating pattern), e.g., repeat units of 2, 3,4, 5, 6, 7, 8, 9, 10, 12, 15, 20, etc., or more droplets. For instance,FIG. 4A illustrates an embodiment where two adjacent droplets areinjected with a dye, followed by three adjacent droplets not injectedwith a dye, thereby creating a 5-droplet repeat unit; in FIG. 4B, a15-droplet repeat unit is illustrated.

In addition, more complex sequences of droplets may be created in achannel, according to certain embodiments, by using more than two typesof droplets. For instance, a channel may contain multiple sites ofinjection, e.g., as previously discussed, and injections may becontrolled to create a first plurality of droplets of a first droplettype, a second plurality of droplets of a second droplet type, and athird plurality of droplets of a third droplet type. Even more droplettypes may be created in other embodiments, e.g., fourth, fifth, sixth,etc. The droplets may be arranged in any suitable pattern, includingarbitrary, random, or repeating patterns (which may have any number ofdroplets defining the repeat unit of the repeating pattern).

In another aspect, the pressure within a channel may be determined usinga system such as those described herein. For example, by determining thebalance of fluids between a first channel and a second channel, thepressure or pressure difference between the channels may be determined.The size or shape of the fluid interface between a first fluid in afirst channel and a second fluid in a second channel is a function, atleast in part, of the difference in pressure between the channels. Bydetermining a property of the interface, e.g., by determining a radiusof curvature of the interface, pressures within the channels on eitherside of the interface may be determined.

As a specific example, a first channel and a second channel mayintersect at an intersection. The first channel may contain a first,continuous liquid. When fluid from the second channel enters the firstchannel at the intersection, a fluidic interface may be formed. Thepressure within the continuous phase may fluctuate due to the partial orcomplete plugging of the first channel by the fluid emerging from thesecond channel. To measure the pressure fluctuations, a sensor such as aLaplace sensor may be positioned in the first channel upstream of theintersection, e.g., as shown in FIG. 13 with first channel 800, secondchannel 810, and sensor 815. Second channel 810 in this example is usedto inject a fluid into first channel 800, in this case creating a newdroplet 820 within channel 810. Early in the cycle, the emerging dropletat least partially plugs first channel 800, causing the pressure in thefirst channel to rise and interface 840 of sensor 815 to move upward, asshown in FIG. 13A. However, when the droplet 820 separates from thesecond channel, the pressure is released, causing interface 840 to movelower in sensor 815, as shown in FIG. 13B. Because the droplets producedvia the second channel are produced in the first channel in a periodiccycle, the interface of the sensor oscillates up and down periodically.By tracking the position of the interface over many cycles, pressurefluctuations within the first channel may be determined or quantifiedduring droplet formation. An example of such a determination can be seenin Example 4.

For example, early in the cycle, when the emerging droplet partially orcompletely plugs the first channel, there is a general ramping up ofpressure within the first channel upstream of the intersection, asregistered by the Laplace sensor. After plugging the orifice at theintersection of the first and second channels, the increased pressureimpinges on the emerging droplet, causing the droplet to narrow andeventually pinch off to form an isolated droplet; this event maycoincide with a rapid fall in pressure.

In various aspects of the invention, a fluidic system as disclosedherein may also include a droplet formation system, a sensing system, acontroller, and/or a droplet sorting and/or separation system, or anycombination of these systems. Such systems and methods may be positionedin any suitable order, depending on a particular application, and insome cases, multiple systems of a given type may be used, for example,two or more droplet formation systems, two or more droplet separationsystems, etc. As examples of arrangements, systems of the invention canbe arranged to form droplets, to dilute fluids, to control theconcentration of species within droplets, to sort droplets to selectthose with a desired concentration of species or entities (e.g.,droplets each containing one molecule of reactant), to fuse individualdroplets to cause reaction between species contained in the individualdroplets, to determine reaction(s) and/or rates of reaction(s) in one ormore droplets, etc. Many other arrangements can be practiced inaccordance with the invention.

One aspect of the invention relates to systems and methods for producingdroplets of fluid surrounded by a liquid. The fluid and the liquid maybe substantially immiscible in many cases, i.e., immiscible on a timescale of interest (e.g., the time it takes a fluidic droplet to betransported through a particular system or device). In certain cases,the droplets may each be substantially the same shape or size, asfurther described below. The fluid may also contain other species, forexample, certain molecular species (e.g., as further discussed below),cells, particles, etc.

In one set of embodiments, electric charge may be created on a fluidsurrounded by a liquid, which may cause the fluid to separate intoindividual droplets within the liquid. In some embodiments, the fluidand the liquid may be present in a channel, e.g., a microfluidicchannel, or other constricted space that facilitates application of anelectric field to the fluid (which may be “AC” or alternating current,“DC” or direct current etc.), for example, by limiting movement of thefluid with respect to the liquid. Thus, the fluid can be present as aseries of individual charged and/or electrically inducible dropletswithin the liquid. In one embodiment, the electric force exerted on thefluidic droplet may be large enough to cause the droplet to move withinthe liquid. In some cases, the electric force exerted on the fluidicdroplet may be used to direct a desired motion of the droplet within theliquid, for example, to or within a channel or a microfluidic channel(e.g., as further described herein), etc.

Electric charge may be created in the fluid within the liquid using anysuitable technique, for example, by placing the fluid within an electricfield (which may be AC, DC, etc.), and/or causing a reaction to occurthat causes the fluid to have an electric charge, for example, achemical reaction, an ionic reaction, a photocatalyzed reaction, etc. Inone embodiment, the fluid is an electrical conductor. As used herein, a“conductor” is a material having a conductivity of at least about theconductivity of 18 megaohm (MOhm or MΩ) water. The liquid surroundingthe fluid may have a conductivity less than that of the fluid. Forinstance, the liquid may be an insulator, relative to the fluid, or atleast a “leaky insulator,” i.e., the liquid is able to at leastpartially electrically insulate the fluid for at least a short period oftime. Those of ordinary skill in the art will be able to identify theconductivity of fluids. In one non-limiting embodiment, the fluid may besubstantially hydrophilic, and the liquid surrounding the fluid may besubstantially hydrophobic.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used. In certain embodiments, the electricfield generator can be constructed and arranged (e.g., positioned) tocreate an electric field applicable to the fluid of at least about 0.01V/micrometer, and, in some cases, at least about 0.03 V/micrometer, atleast about 0.05 V/micrometer, at least about 0.08 V/micrometer, atleast about 0.1 V/micrometer, at least about 0.3 V/micrometer, at leastabout 0.5 V/micrometer, at least about 0.7 V/micrometer, at least about1 V/micrometer, at least about 1.2 V/micrometer, at least about 1.4V/micrometer, at least about 1.6 V/micrometer, or at least about 2V/micrometer. In some embodiments, even higher electric fieldintensities may be used, for example, at least about 2 V/micrometer, atleast about 3 V/micrometer, at least about 5 V/micrometer, at leastabout 7 V/micrometer, or at least about 10 V/micrometer or more.

In another aspect, the invention relates to systems and methods forallowing the mixing of more than one fluid to occur. For example, invarious embodiments of the invention, two or more fluidic droplets maybe allowed to fuse or coalesce, as described above, and then, within thefused droplet, the two or more fluids from the two or more originalfluidic droplets may then be allowed to mix. It should be noted thatwhen two droplets fuse or coalesce, perfect mixing within the dropletdoes not instantaneously occur. Instead, for example, the coalesceddroplet may initially be formed of a first fluid region and a secondfluid region. In some cases, the fluid regions may remain as separateregions, for example, due to internal “counter-revolutionary” flowwithin the fluidic droplet, thus resulting in a non-uniform fluidicdroplet

However, in other cases, the fluid regions within the fluidic dropletmay be allowed to mix, react, or otherwise interact with each other,resulting in mixed or partially mixed fluidic droplets. The mixing mayoccur through natural means, for example, through diffusion (e.g.,through the interface between the regions), through reaction of thefluids with each other, through fluid flow within the droplet (i.e.,convection), etc. However, in some cases, mixing of the regions may beenhanced through certain systems external of the fluidic droplet. Forexample, the fluidic droplet may be passed through one or more channelsor other systems which cause the droplet to change its velocity and/ordirection of movement. The change of direction may alter convectionpatterns within the droplet, causing the fluids to be at least partiallymixed.

Other examples of fluidic mixing in droplets are described inInternational Patent Application Serial No. PCT/US2004/010903, filedApr. 9, 2004 by Link, et al., incorporated herein by reference.

In another set of embodiments, droplets of fluid can be created from afluid surrounded by a liquid within a channel by altering the channeldimensions in a manner that is able to induce the fluid to formindividual droplets, for example, using flow-focusing techniques. Thechannel may, for example, be a channel that expands relative to thedirection of flow, e.g., such that the fluid does not adhere to thechannel walls and forms individual droplets instead, or a channel thatnarrows relative to the direction of flow, e.g., such that the fluid isforced to coalesce into individual droplets. In other embodiments,internal obstructions may also be used to cause droplet formation tooccur. For instance, baffles, ridges, posts, or the like may be used todisrupt liquid flow in a manner that causes the fluid to coalesce intofluidic droplets.

Other examples of the production of droplets of fluid surrounded by aliquid are described in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al. and InternationalPatent Application Serial No. PCT/US03/20542, filed Jun. 30, 2003 byStone, et al., published as WO 2004/002627 on Jan. 8, 2004, eachincorporated herein by reference.

In some embodiments, the fluidic droplets may each be substantially thesame shape and/or size. The shape and/or size can be determined, forexample, by measuring the average diameter or other characteristicdimension of the droplets. The term “determining,” as used herein,generally refers to the analysis or measurement of a species, forexample, quantitatively or qualitatively, and/or the detection of thepresence or absence of the species. “Determining” may also refer to theanalysis or measurement of an interaction between two or more species,for example, quantitatively or qualitatively, or by detecting thepresence or absence of the interaction. Examples of suitable techniquesinclude, but are not limited to, spectroscopy such as infrared,absorption, fluorescence, UV/visible, FTIR (“Fourier Transform InfraredSpectroscopy”), or Raman; gravimetric techniques; ellipsometry;piezoelectric measurements; immunoassays; electrochemical measurements;optical measurements such as optical density measurements; circulardichroism; light scattering measurements such as quasielectric lightscattering; polarimetry; refractometry; or turbidity measurements.

The “average diameter” of a plurality or series of droplets is thearithmetic average of the average diameters of each of the droplets.Those of ordinary skill in the art will be able to determine the averagediameter (or other characteristic dimension) of a plurality or series ofdroplets, for example, using laser light scattering, microscopicexamination, or other known techniques. The diameter of a droplet, in anon-spherical droplet, is the mathematically-defined average diameter ofthe droplet, integrated across the entire surface. The average diameterof a droplet (and/or of a plurality or series of droplets) may be, forexample, less than about 1 mm, less than about 500 micrometers, lessthan about 200 micrometers, less than about 100 micrometers, less thanabout 75 micrometers, less than about 50 micrometers, less than about 25micrometers, less than about 10 micrometers, or less than about 5micrometers in some cases. The average diameter may also be at leastabout 1 micrometer, at least about 2 micrometers, at least about 3micrometers, at least about 5 micrometers, at least about 10micrometers, at least about 15 micrometers, or at least about 20micrometers in certain cases.

In certain embodiments of the invention, the fluidic droplets maycontain additional entities, for example, other chemical, biochemical,or biological entities (e.g., dissolved or suspended in the fluid),cells, particles, gases, molecules, or the like. In some cases, thedroplets may each be substantially the same shape or size, as discussedabove. In certain instances, the invention provides for the productionof droplets consisting essentially of a substantially uniform number ofentities of a species therein (i.e., molecules, cells, particles, etc.).For example, about 90%, about 93%, about 95%, about 97%, about 98%, orabout 99%, or more of a plurality or series of droplets may each containthe same number of entities of a particular species. For instance, asubstantial number of fluidic droplets produced, e.g., as describedabove, may each contain 1 entity, 2 entities, 3 entities, 4 entities, 5entities, 7 entities, 10 entities, 15 entities, 20 entities, 25entities, 30 entities, 40 entities, 50 entities, 60 entities, 70entities, 80 entities, 90 entities, 100 entities, etc., where theentities are molecules or macromolecules, cells, particles, etc. In somecases, the droplets may each independently contain a range of entities,for example, less than 20 entities, less than 15 entities, less than 10entities, less than 7 entities, less than 5 entities, or less than 3entities in some cases. In one set of embodiments, in a liquidcontaining droplets of fluid, some of which contain a species ofinterest and some of which do not contain the species of interest, thedroplets of fluid may be screened or sorted for those droplets of fluidcontaining the species as further described below (e.g., usingfluorescence or other techniques such as those described above), and insome cases, the droplets may be screened or sorted for those droplets offluid containing a particular number or range of entities of the speciesof interest, e.g., as previously described.

In another aspect, the invention relates to systems and methods forsplitting a fluidic droplet into two or more droplets. The fluidicdroplet may be surrounded by a liquid, e.g., as previously described,and the fluid and the liquid are essentially immiscible in some cases.The two or more droplets created by splitting the original fluidicdroplet may each be substantially the same shape and/or size, or the twoor more droplets may have different shapes and/or sizes, depending onthe conditions used to split the original fluidic droplet.

According to one set of embodiments, a fluidic droplet can be splitusing an applied electric field. The electric field may be an AC field,a DC field, etc. The fluidic droplet, in this embodiment, may have agreater electrical conductivity than the surrounding liquid, and, insome cases, the fluidic droplet may be neutrally charged. In someembodiments, the droplets produced from the original fluidic droplet areof approximately equal shape and/or size. In certain embodiments, in anapplied electric field, electric charge may be urged to migrate from theinterior of the fluidic droplet to the surface to be distributedthereon, which may thereby cancel the electric field experienced in theinterior of the droplet. In some embodiments, the electric charge on thesurface of the fluidic droplet may also experience a force due to theapplied electric field, which causes charges having opposite polaritiesto migrate in opposite directions. The charge migration may, in somecases, cause the drop to be pulled apart into two separate fluidicdroplets. The electric field applied to the fluidic droplets may becreated, for example, using the techniques described above, such as witha reaction an electric field generator, etc.

Other examples of splitting a fluidic droplet into two droplets aredescribed in International Patent Application Serial No.PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; U.S. ProvisionalPatent Application Ser. No. 60/498,091, filed Aug. 27, 2003, by Link,et. al.; and International Patent Application Serial No. PCT/US03/20542,filed Jun. 30, 2003 by Stone, et al., published as WO 2004/002627 onJan. 8, 2004, each incorporated herein by reference.

In one set of embodiments, a fluidic droplet may be directed by creatingan electric charge (e.g., as previously described) on the droplet, andsteering the droplet using an applied electric field, which may be an ACfield, a DC field, etc. As an example, an electric field may beselectively applied and removed (or a different electric field may beapplied) as needed to direct the fluidic droplet to a particular region.The electric field may be selectively applied and removed as needed, insome embodiments, without substantially altering the flow of the liquidcontaining the fluidic droplet.

In other embodiments, however, the fluidic droplets may be screened orsorted within a fluidic system of the invention by altering the flow ofthe liquid containing the droplets. For instance, in one set ofembodiments, a fluidic droplet may be steered or sorted by directing theliquid surrounding the fluidic droplet into a first channel, a secondchannel, etc.

In certain aspects of the invention, sensors are provided that can senseand/or determine one or more characteristics of the fluidic droplets,and/or a characteristic of a portion of the fluidic system containingthe fluidic droplet (e.g., the liquid surrounding the fluidic droplet)in such a manner as to allow the determination of one or morecharacteristics of the fluidic droplets. Characteristics determinablewith respect to the droplet and usable in the invention can beidentified by those of ordinary skill in the art. Non-limiting examplesof such characteristics include fluorescence, spectroscopy (e.g.,optical, infrared, ultraviolet, etc.), radioactivity, mass, volume,density, temperature, viscosity, pH, concentration of a substance, suchas a biological substance (e.g., a protein, a nucleic acid, etc.), orthe like.

A variety of definitions are now provided which will aid inunderstanding various aspects of the invention. Following, andinterspersed with these definitions, is further disclosure that willmore fully describe the invention.

As noted, various aspects of the present invention relate to droplets offluid surrounded by a liquid (e.g., suspended). The droplets may be ofsubstantially the same shape and/or size, or of different shapes and/orsizes, depending on the particular application. As used herein, the term“fluid” generally refers to a substance that tends to flow and toconform to the outline of its container, i.e., a liquid, a gas, aviscoelastic fluid, etc. Typically, fluids are materials that are unableto withstand a static shear stress, and when a shear stress is applied,the fluid experiences a continuing and permanent distortion. The fluidmay have any suitable viscosity that permits flow. If two or more fluidsare present, each fluid may be independently selected among essentiallyany fluids (liquids, gases, and the like) by those of ordinary skill inthe art, by considering the relationship between the fluids. The fluidsmay each be miscible or immiscible. For example, two fluids can beselected to be essentially immiscible within the time frame of formationof a stream of fluids, or within the time frame of reaction orinteraction. Where the portions remain liquid for a significant periodof time, then the fluids should be essentially immiscible. Where, aftercontact and/or formation, the dispersed portions are quickly hardened bypolymerization or the like, the fluids need not be as immiscible. Thoseof ordinary skill in the art can select suitable miscible or immisciblefluids, using contact angle measurements or the like, to carry out thetechniques of the invention.

As used herein, a first entity is “surrounded” by a second entity if aclosed planar loop can be drawn around the first entity through only thesecond entity. A first entity is “completely surrounded” if closed loopsgoing through only the second entity can be drawn around the firstentity regardless of direction (orientation of the loop). In oneembodiment, the first entity is a cell, for example, a cell suspended inmedia is surrounded by the media. In another embodiment, the firstentity is a particle. In yet another embodiment, the first entity is afluid. The second entity may also be a fluid in some cases (e.g., as ina suspension, an emulsion, etc.), for example, a hydrophilic liquid maybe suspended in a hydrophobic liquid, a hydrophobic liquid may besuspended in a hydrophilic liquid, a gas bubble may be suspended in aliquid, etc. Typically, a hydrophobic liquid and a hydrophilic liquidare essentially immiscible with respect to each other, where thehydrophilic liquid has a greater affinity to water than does thehydrophobic liquid. Examples of hydrophilic liquids include, but are notlimited to, water and other aqueous solutions comprising water, such ascell or biological media, salt solutions, etc., as well as otherhydrophilic liquids such as ethanol. Examples of hydrophobic liquidsinclude, but are not limited to, oils such as hydrocarbons, siliconeoils, mineral oils, fluorocarbon oils, organic solvents etc. Otherexamples of suitable fluids have been previously described.

Similarly, a “droplet,” as used herein, is an isolated portion of afirst fluid that is completely surrounded by a second fluid. It is to benoted that a droplet is not necessarily spherical, but may assume othershapes as well, for example, depending on the external environment. Inone embodiment, the droplet has a minimum cross-sectional dimension thatis substantially equal to the largest dimension of the channelperpendicular to fluid flow in which the droplet is located.

As mentioned, in some, but not all embodiments, the systems and methodsdescribed herein may include one or more microfluidic components, forexample, one or more microfluidic channels. “Microfluidic,” as usedherein, refers to a device, apparatus or system including at least onefluid channel having a cross-sectional dimension of less than 1 mm, anda ratio of length to largest cross-sectional dimension of at least 3:1.A “microfluidic channel,” as used herein, is a channel meeting thesecriteria. The “cross-sectional dimension” of the channel is measuredperpendicular to the direction of fluid flow within the channel. Thus,some or all of the fluid channels in microfluidic embodiments of theinvention may have maximum cross-sectional dimensions less than 2 mm,and in certain cases, less than 1 mm. In one set of embodiments, allfluid channels containing embodiments of the invention are microfluidicor have a largest cross sectional dimension of no more than 2 mm or 1mm. In certain embodiments, the fluid channels may be formed in part bya single component (e.g. an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids and/or deliver fluids to various components or systems of theinvention. In one set of embodiments, the maximum cross-sectionaldimension of the channel(s) containing embodiments of the invention isless than 500 microns, less than 200 microns, less than 100 microns,less than 50 microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs flow of a fluid. The channelcan have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and/or outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, 10:1,15:1, 20:1, or more. An open channel generally will includecharacteristics that facilitate control over fluid transport, e.g.,structural characteristics (an elongated indentation) and/or physical orchemical characteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

In one set of embodiments, the fluidic droplets may contain cells orother entities, such as proteins, viruses, macromolecules, particles,etc. As used herein, a “cell” is given its ordinary meaning as used inbiology. The cell may be any cell or cell type. For example, the cellmay be a bacterium or other single-cell organism, a plant cell, or ananimal cell.

A variety of materials and methods, according to certain aspects of theinvention, can be used to form any of the above-described components ofthe systems and devices of the invention. In some cases, the variousmaterials selected lend themselves to various methods. For example,various components of the invention can be formed from solid materials,in which the channels can be formed via micromachining, film depositionprocesses such as spin coating and chemical vapor deposition, laserfabrication, photolithographic techniques, etching methods including wetchemical or plasma processes, and the like. See, for example, ScientificAmerican, 248:44-55, 1983 (Angell, et al). In one embodiment, at least aportion of the fluidic system is formed of silicon by etching featuresin a silicon chip. In some cases, one or more microfluidic channels maybe defined in a solid material, and in some cases, various microfluidicchannels may be defined in separate solid or planar materials that arephysically contacted together. For instance, a first planar material maycontain a first fluidic channel, and a second planar material maycontain a second fluidic channel, where the materials are physicallycontacted together and an orifice defined between the first and secondplanar materials such that fluid can flow from the second channel intothe first channel, e.g., through an orifice, using the systems andmethods discussed above. Technologies for precise and efficientfabrication of various fluidic systems and devices of the invention fromsilicon are known. In another embodiment, various components of thesystems and devices of the invention can be formed of a polymer, forexample, an elastomeric polymer such as polydimethylsiloxane (“PDMS”),polytetrafluoroethylene (“PTFE” or Teflon®), or the like.

Different components can be fabricated of different materials. Forexample, a base portion including a bottom wall and side walls can befabricated from an opaque material such as silicon or PDMS, and a topportion can be fabricated from a transparent or at least partiallytransparent material, such as glass or a transparent polymer, forobservation and/or control of the fluidic process. Components can becoated so as to expose a desired chemical functionality to fluids thatcontact interior channel walls, where the base supporting material doesnot have a precise, desired functionality. For example, components canbe fabricated as illustrated, with interior channel walls coated withanother material. Material used to fabricate various components of thesystems and devices of the invention, e.g., materials used to coatinterior walls of fluid channels, may desirably be selected from amongthose materials that will not adversely affect or be affected by fluidflowing through the fluidic system, e.g., material(s) that is chemicallyinert in the presence of fluids to be used within the device.

In one embodiment, various components of the invention are fabricatedfrom polymeric and/or flexible and/or elastomeric materials, and can beconveniently formed of a hardenable fluid, facilitating fabrication viamolding (e.g. replica molding, injection molding, cast molding, etc.).The hardenable fluid can be essentially any fluid that can be induced tosolidify, or that spontaneously solidifies, into a solid capable ofcontaining and/or transporting fluids contemplated for use in and withthe fluidic network. In one embodiment, the hardenable fluid comprises apolymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).Suitable polymeric liquids can include, for example, thermoplasticpolymers, thermoset polymers, or mixture of such polymers heated abovetheir melting point. As another example, a suitable polymeric liquid mayinclude a solution of one or more polymers in a suitable solvent, whichsolution forms a solid polymeric material upon removal of the solvent,for example, by evaporation. Such polymeric materials, which can besolidified from, for example, a melt state or by solvent evaporation,are well known to those of ordinary skill in the art. A variety ofpolymeric materials, many of which are elastomeric, are suitable, andare also suitable for forming molds or mold masters, for embodimentswhere one or both of the mold masters is composed of an elastomericmaterial. A non-limiting list of examples of such polymers includespolymers of the general classes of silicone polymers, epoxy polymers,and acrylate polymers. Epoxy polymers are characterized by the presenceof a three-membered cyclic ether group commonly referred to as an epoxygroup, 1,2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Non-limiting examples of siliconeelastomers suitable for use according to the invention include thoseformed from precursors including the chlorosilanes such asmethylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example,the silicone elastomer polydimethylsiloxane. Non-limiting examples ofPDMS polymers include those sold under the trademark Sylgard by DowChemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard 184,and Sylgard 186. Silicone polymers including PDMS have severalbeneficial properties simplifying fabrication of the microfluidicstructures of the invention. For instance, such materials areinexpensive, readily available, and can be solidified from aprepolymeric liquid via curing with heat. For example, PDMSs aretypically curable by exposure of the prepolymeric liquid to temperaturesof about, for example, about 65° C. to about 75° C. for exposure timesof, for example, about an hour. Also, silicone polymers, such as PDMS,can be elastomeric and thus may be useful for forming very smallfeatures with relatively high aspect ratios, necessary in certainembodiments of the invention. Flexible (e.g., elastomeric) molds ormasters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures ofthe invention from silicone polymers, such as PDMS, is the ability ofsuch polymers to be oxidized, for example by exposure to anoxygen-containing plasma such as an air plasma, so that the oxidizedstructures contain, at their surface, chemical groups capable ofcross-linking to other oxidized silicone polymer surfaces or to theoxidized surfaces of a variety of other polymeric and non-polymericmaterials. Thus, components can be fabricated and then oxidized andessentially irreversibly sealed to other silicone polymer surfaces, orto the surfaces of other substrates reactive with the oxidized siliconepolymer surfaces, without the need for separate adhesives or othersealing means. In most cases, sealing can be completed simply bycontacting an oxidized silicone surface to another surface without theneed to apply auxiliary pressure to form the seal. That is, thepre-oxidized silicone surface acts as a contact adhesive againstsuitable mating surfaces. Specifically, in addition to beingirreversibly sealable to itself, oxidized silicone such as oxidized PDMScan also be sealed irreversibly to a range of oxidized materials otherthan itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention, as well as overall molding techniques, are described in theart, for example, in an article entitled “Rapid Prototyping ofMicrofluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480,1998 (Duffy et al.), incorporated herein by reference.

Another advantage to forming microfluidic structures of the invention(or interior, fluid-contacting surfaces) from oxidized silicone polymersis that these surfaces can be much more hydrophilic than the surfaces oftypical elastomeric polymers (where a hydrophilic interior surface isdesired). Such hydrophilic channel surfaces can thus be more easilyfilled and wetted with aqueous solutions than can structures comprisedof typical, unoxidized elastomeric polymers or other hydrophobicmaterials.

In one embodiment, a bottom wall is formed of a material different fromone or more side walls or a top wall, or other components. For example,the interior surface of a bottom wall can comprise the surface of asilicon wafer or microchip, or other substrate. Other components can, asdescribed above, be sealed to such alternative substrates. Where it isdesired to seal a component comprising a silicone polymer (e.g. PDMS) toa substrate (bottom wall) of different material, the substrate may beselected from the group of materials to which oxidized silicone polymeris able to irreversibly seal (e.g., glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, andglassy carbon surfaces which have been oxidized). Alternatively, othersealing techniques can be used, as would be apparent to those ofordinary skill in the art, including, but not limited to, the use ofseparate adhesives, thermal bonding, solvent bonding, ultrasonicwelding, etc.

Combinations of these and/or other systems and methods of controllingand manipulating of fluids are also envisioned, for example, systems andmethods as disclosed in U.S. patent application Ser. No. 11/360,845,filed Feb. 23, 2006, by Link, et. al.; U.S. Pat. No. 5,512,131, issuedApr. 30, 1996 to Kumar, et al.; International Patent Publication WO96/29629, published Jun. 26, 1996 by Whitesides, et al.; U.S. Pat. No.6,355,198, issued Mar. 12, 2002 to Kim, et al.; International PatentApplication Serial No.: PCT/US01/16973, filed May 25, 2001 by Anderson,et al., published as WO 01/89787 on Nov. 29, 2001; International PatentApplication Serial No. PCT/US03/20542, filed Jun. 30, 2003 by Stone, etal., published as WO 2004/002627 on Jan. 8, 2004; International PatentApplication Serial No. PCT/US2004/010903, filed Apr. 9, 2004 by Link, etal.; U.S. patent application Ser. No. 11/246,911, filed Oct. 7, 2005, byLink, et al.; and U.S. Patent Application Ser. No. 61/098,674, filedSep. 19, 2008 by Weitz et al., each of which is incorporated herein byreference.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

This example demonstrates fluid injection into fluidic droplets. In eachof Examples 1, 2, and 3, single-layer PDMS microfluidic devices such asthose shown in FIGS. 4A and 4B were fabricated. In these examples,water-in-oil emulsion droplets with relatively low polydispersity flowedpast the orifice where an electric field was applied to cause injectionof a bromophenol dyed fluid into the droplets. The droplets passedthrough a serpentine channel (e.g., as shown in FIGS. 4A and 4B) wherethe bromophenol dye mixed with the fluid inside the droplets. Thedroplets then flowed past a laser beam/PMT (photomultiplier tube) setupand microscope camera, both of which captured the light intensity insidethe droplets. Since the volume of each droplet prior to injection isknown and essentially the same, the amount of bromophenol dyed fluidinjected could be determined from the change in intensity of the dropletafter injection. Measurements from the PMT/laser were compared to imagescaptured by the camera to ensure consistency. Approximately 1,000droplets were sampled for each data point. An example of such anexperiment is shown in FIG. 6. In this figure, the number sizedistribution indicates that fluid was injected into the droplets with arelatively narrow distribution.

Example 2

This example demonstrates control of fluid injection by varying thecontinuous phase (i.e., the “outer phase”) flow rate. A system similarto that discussed in Example 1 was used for this example. The outerphase flow rate was set by a volume-driven syringe pump and was variedwhile keeping the other operating conditions (i.e., applied voltage)constant. As shown in FIG. 7A, as the outer phase flow rate increased,the residence time of droplets decreased, resulting in a decrease in thevolume of fluid injected. Thus, by controlling the outer phase flowrate, the amount of fluid injected into the droplets could be readilycontrolled.

Example 3

This example demonstrates control of fluid injection by varying theinjection pressure. A system similar to that discussed in Example 1 wasused for this example. The fluid tubing leading into the second channelcontaining a bromophenol dye was placed in a pressure tight basin fed byregulator-controlled pressurized air. As the injection pressureincreased, the injection volume increased, as shown in FIG. 7B (pressurein units of ps). Accordingly, by controlling the injection pressure, theamount of fluid injected into the droplets could be readily controlled.

Example 4

This example illustrates the determination of pressure in a fluidicchannel locally and with high temporal resolution, in another embodimentof the invention.

FIG. 12 illustrates the Laplace sensors used in this example to measurechannel pressure locally and with high temporal resolution. In FIG. 12A,first channel 850 and second channel 860 meet at intersection 870.However, prior to intersection 870, first channel 850 and second channel860 run substantially parallel to each other. Second channel ends atorifice 865 at intersection 870. First channel 850 contains a first,outer fluid while second channel 860 contains a second, inner fluid (inthese figures, the fluids appear as different colors, and the terms“inner” and “outer” are used for purposes of clarity only). The fluidicinterface between these fluids is balanced at the orifice by adjustingthe pressures of the two fluids. By controlling the pressures within thefirst and second channels, as is shown in FIG. 12B, the interfacebetween the two fluids may be controlled to adopt a shape with fixedcurvature such that the pressure jump across the interface is equal tothe pressure differential inside and outside. In FIG. 12C, by measuringthe distance of the interface below the plane of the orifice, the radiusof curvature of the bulge can be calculated. Using Laplace's Law, thiscan then be used to calculate the pressure in a channel, e.g., using anoptical measurement of the location of the interface.

The following is an explanation of how the location of the interface canbe used to calculate the pressure in a channel. However, it should beunderstood that this discussion is not meant to be limiting. When theinterface between the two fluids is balanced in the orifice, thepressure differential between the inner (second) and outer (first)fluids is equal to the Laplace pressure jump across the interface,

P _(out) −P _(in) =γ/r.

For fixed P_(in), the radius of curvature of the interface r depends onP_(out): If P_(out) is small, the interface is positioned relatively lowin the orifice, adopting a shape of high curvature, whereas if it islarge, it is positioned relatively high in the orifice, adopting aflatter shape with lower curvature. If the pressure fluctuates in time,the interface may move to maintain mechanical equilibrium in eachinstant. Thus, the value of r(t) can be used to indirectly calculateP_(out)(t) at each time point. To obtain r(t), h(t) can be determined,where h(t) is the distance of the lowest part of the interface from theplane of the orifice, as shown in FIG. 12. For example, for a circularorifice,

r(t)=½[h(t)+d ² /h(t)].

Since P_(out)(t)=γ/r(t)+P_(in), this allows the pressure fluctuations tobe determined by optically tracking the motion of the interface.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of”, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-27. (canceled)
 28. A method, comprising: providinga microfluidic system comprising a first microfluidic channel and asecond microfluidic channel contacting the first microfluidic channel atan intersection; providing a first fluid in the first microfluidicchannel and a second fluid in the second microfluidic channel, whereinthe first fluid and the second fluid contact each other at leastpartially within the intersection to define a fluidic interface; andurging the second fluid to enter the first microfluidic channel,wherein, when an electric field is applied to the interface, the secondfluid is at least partially prevented from entering the firstmicrofluidic channel.
 29. The method of claim 28, wherein the firstfluid is present within the first microfluidic channel as a plurality ofdroplets contained within a carrying fluid.
 30. The method of claim 28,wherein the second droplet is urged to form droplets within the firstmicrofluidic channel in the absence of the electric field is applied.31. The method of claim 28, wherein the second droplet is urged into adroplet of the first fluid contained within a carrying fluid containedwithin the first microfluidic channel.
 32. The method of claim 31,wherein the droplet of the first fluid is completely surrounded by thecarrying fluid.
 33. The method of claim 28, wherein the firstmicrofluidic channel contains an inlet portion and an outlet portionrelative to the intersection for carrying the first fluid.
 34. Themethod of claim 28, wherein the second microfluidic channel containsonly an inlet portion relative to the intersection for carrying thesecond fluid.
 35. A method, comprising: providing a microfluidic systemcomprising a first microfluidic channel and a second microfluidicchannel contacting the first microfluidic channel at an intersection;providing a first fluid in the first microfluidic channel and a secondfluid in the second microfluidic channel, wherein the first fluid andthe second fluid contact each other at least partially within theintersection to define a fluidic interface; and urging fluid from thefirst microfluidic channel into the second microfluidic channel,wherein, when an electric field is applied to the interface, the fluidis at least partially prevented from entering the second microfluidicchannel.
 36. The method of claim 35, wherein the first fluid is presentwithin the first microfluidic channel as a plurality of dropletscontained within a carrying fluid.
 37. The method of claim 35, whereinthe second droplet is urged to form droplets within the firstmicrofluidic channel in the absence of the electric field is applied.38. The method of claim 35, wherein the second droplet is urged into adroplet of the first fluid contained within a carrying fluid containedwithin the first microfluidic channel.
 39. The method of claim 35,wherein the droplet of the first fluid is completely surrounded by thecarrying fluid.
 40. The method of claim 35, wherein the firstmicrofluidic channel contains an inlet portion and an outlet portionrelative to the intersection for carrying the first fluid.
 41. Themethod of claim 35, wherein the second microfluidic channel containsonly an inlet portion relative to the intersection for carrying thesecond fluid.